1. Field of the Invention
The present invention relates to a laser scanning microscope for scanning a laser beam onto a sample to detect fluorescence from the sample by a photodetector.
2. Description of the Related Art
In Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, a laser scanning microscope is disclosed including a first optical scanning system A for obtaining a scanned image of fluorescence from the sample and a second optical scanning system B for expressing peculiar phenomena such as cleavage of a caged reagent in a specific portion of the sample.
FIG. 1 is a diagram showing a constitution of a conventional laser scanning microscope. A sample 79 is irradiated with laser beams from the second optical scanning system B in synchronization with the scanning of the laser beams of the first optical scanning system A, and changes of the sample 79 with an elapse of time can be measured. The synchronization is carried out, when a control unit 81 controls a laser shutter 63, optical scanning unit 64, and photoelectric conversion device 70 of the first optical scanning system A, and a laser shutter 72 and optical scanning unit 73 of the second optical scanning system B.
The caged reagent and a fluorescent indicator having sensitivity to concentration of ions such as calcium ions are injected into the sample 79. The sample 79 in which the caged reagent has been injected is irradiated with the laser beams from a laser unit 71 of the second optical scanning system B. A caged group of the caged reagent in the irradiated portion is cloven, and materials enclosed inside are released. The change of an ion concentration distribution in the sample 79 by this release is measured by a fluorescent image obtained by the laser beams from a laser unit 61 of the first optical scanning system A. With the cleavage of the caged reagent or by the irradiation with the laser beams of the second laser unit 71, the fluorescent indicator of the sample 79 produces a certain degree of fluorescence. However, the control unit 81 controls an opening/closing timing of the laser shutters 63, 72 of each laser beam and a detection timing in the photoelectric conversion device 70 with the elapse of time. Therefore, a spectrum of fluorescence can be detected by a photodetector to obtain the fluorescent image without being influenced by the change of a fluorescent intensity from the fluorescent indicator with the cleavage of the caged reagent.
However, in the laser scanning microscope including first and second optical scanning systems described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, there is possibility that the laser beams of the second optical scanning system are detected by the photodetector of the first optical scanning system. This has left much room for improvement in obtaining a desired fluorescent image.
For example, the use of a UV pulse laser (wavelength of 351 nm) as the laser unit 71 of the second optical scanning system B for cleaving the caged reagent is considered. Since much light intensity is required for cleaving the caged reagent, a reflected light of the laser beams of the second optical scanning system from the irradiated sample 79 is also intense. A dichroic mirror 75 does not sufficiently absorb the reflected light of the UV pulse laser beams, and a slight amount of the light is transmitted through an optical path of the first optical scanning system A. However, in a dichroic mirror 62 and filters such as a laser cut filter 67 usually for use in the first optical scanning system A, that is, an optical scanning system for acquiring images, transmission capabilities with respect to a short wavelength band of the UV laser are hardly considered. The wavelength of the UV pulse laser is reflected, transmitted, and detected by the photoelectric conversion device 70, and a clear fluorescent image cannot be obtained.
Similarly, the use of an IR pulse laser (wavelength of 710 nm) as the laser unit 71 of the second optical scanning system B for cleaving the caged reagent is considered. It is to be noted that this IR pulse laser is assumed as laser capable of causing two photon excitation. Also for the IR pulse laser, the intense reflected light from the sample 79 is not sufficiently reflected by the dichroic mirror 75, and the slight amount of the light passes through the optical path of the first optical scanning system A. For the filters usually for use in the first optical scanning system A, that is, the optical scanning system for acquiring the images, a long path filter which reflects a short wavelength and transmits a long wavelength is used in many cases. For these laser cut filters, transmission characteristics in the long wavelength band of IR are not considered. Therefore, the wavelength of IR pulse laser beams, which is longer than that of the fluorescence, passes through the laser cut filter, and is detected by the photodetector. Therefore, the clear fluorescent image cannot be obtained.
Moreover, to prevent the above-described phenomenon, as described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, it is considered that the control unit 81, for example, shifts a timing of laser irradiation to control the first and second optical scanning systems, and influences of the laser beams of the second optical scanning system B are avoided. However, in this case, since it is necessary to simultaneously control the optical scanning system and an optical detection system at a high speed, a complicate control is required for realizing this. Furthermore, in the technique described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-275529, the sample cannot be irradiated with two types of laser beams at the same time. Therefore, when the changes of the sample 79 with the elapse of time are measured, real time characteristics drop.